We have recently shown that glucose-dependent insulinotropic polypeptide (GIP)-dependent primary bilateral macronodular adrenal hyperplasia (PBMAH) with Cushing’s syndrome, is a genetic disease caused by germline inactivating mutations in lysine demethylase 1A (KDM1A or LSD1) with loss of heterozygosity of the second KDM1A locus in adrenal lesions, which occurs almost exclusively in women. KDM1A promotes a refractory chromatin state to gene expression by demethylating histone H3 on lysine 4 and can also activate gene transcription by demethylating histone H3 on lysine 9, or by interacting with transcription factors. We hypothesize that loss of KDM1A promotes the development of adrenal hyperplasia by activating the expression of sexually dimorphic genes involved in adrenocortical cell proliferation through chromatin remodeling associated with the loss of repressive histone marks. In mice, Kdm1a is required for pituitary organogenesis and pancreatic endocrine cell development. We therefore hypothesize that KDM1A is also required for the transition from fetal to adult adrenal cortex that occurs specifically in primates. Kdm1a is a master controller of gene transcription in spermatogonia and is essential for the maintenance and differentiation of spermatogonial stem cells in mice, but its involvement in (later stages of) spermatogenesis in men is unknown. We hypothesize, that the absence of functional KDM1A occurring during meiosis in 50% of spermatozoids in KDM1A-mutation carriers alters their H3K4 methylation marks and other sperm histones. Our objectives are to: 1) to decipher the molecular mechanism(s) by which loss of KDM1A promotes adrenal hyperplasia and investigate the underpinnings of the sex bias in this disease, with a special focus on the potential misexpression of the X-linked genes at single-cell resolution. 2) to study the expression and function of KDM1A and GIPR during adrenal cortex development in cortex of cynomolgus macaques, by performing repeated echo-guided adrenal gland biopsies in utero in 4 fetuses at 18 weeks gestational age, and further postnatally at 1-2 week and at 6 months of life. Prior to these biopsies, we will investigate the cortisol response to GIP injection in vivo. 3) to investigate adrenal function and morphology in apparently healthy carriers of KDM1A mutation and analyze histone H3 methylation in their sperm. We plan to genotype > 120 family members of patients with GIP-dependent PBMAH from our cohort, and to perform biochemical screening and adrenal CT scans in KDM1A-mutation carriers to describe the prevalence and the natural history of adrenal hyperplasia and multiple myeloma. If sperm from KDM1A-mutation carriers show altered H3K4 methylation, we will attempt to link the observed variations to specific genomic loci. This project is consistent with governmental plans and strategic research priorities for 2023, as it develops “Translational research in rare diseases”. The use of complementary experimental approaches including studies on adrenal samples derived from patients, cell lines, mouse models, and non-human primates, as well as clinical investigations in patients, illustrates the translational nature of the project. Our project will provide new insights into a key question in cell biology and medicine: how (adrenal) cells maintain a specific transcriptomic pattern that defines their identity during development and in pathological situations, focusing on KDM1A as an epigenetic regulator of cell fate determination. Genetic and biochemical screening in families of patients with KDM1A mutation is important, as it will allow for earlier detection of adrenal hyperplasia and prevent the insidious effects of progressive cortisol excess on health. The feasibility of the project is underlined by published and preliminary results and the complementarity of the consortium members, who are renowned researchers in their field.